title: Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase from pseudomonas sp. strain P.J. 874.
abstract: Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) from Pseudomonas sp. strain P.J. 874 were studied with 14C- and different 3H-labelled 4-hydroxyphenylpyruvate. Tritium of ring-2,6-3H2-labelled substrate was released into water in 1:2 stoichiometry to 14CO2 formation. The tritium release from ring-3,5-3H2- and side chain-3-3H1-labelled 4-hydroxyphenylpyruvate was low as compared with 14CO2 formation. The apparent tritium isotope effects were below two, as judged by comparison of 3H/14C ratios of 4-hydroxyphenylpyruvate and homogentisate. The ratios showed no dependence on oxygen concentrations between 1 and 21% in the gas phase. Thus, a tritium assay can be used to determine the activity of 4-hydroxyphenylpyruvate dioxygenase. Apparently, none of the substrate hydrogens is involved in any rate-limiting step up to the first irreversible step. enol-4-Hydroxyphenylpyruvate was excluded as the active substrate tautomer.
mesh_terms: D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism; D002250:Carbon Radioisotopes; D010105:Oxygenases / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology; D013379:Substrate Specificity; D014316:Tritium / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D014316:Tritium; D002245:Carbon Dioxide; D010105:Oxygenases; D015111:4-Hydroxyphenylpyruvate Dioxygenase